Antiviral and antibacteria agents based on quaternary ammonium compound complexed with boric acid and its derivatives

ABSTRACT

The present document describes compounds resulting from the complexation of quaternary ammonium compounds with boric acid and/or its derivatives, and methods of making the same, and methods of using the same for the treatment of pathogenic infections.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (SEQ.txt; Size: 4,657bytes; and Date of Creation: Oct. 16, 2017) is herein incorporated byreference in its entirety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 15/566,766, filed Oct. 14, 2017, which is a USNational Phase under 35 USC § 371 of PCT/CA2016/050431, filed Apr. 14,2016, which claims priority from and the benefit of U.S. ProvisionalPatent Application 62/147,161 filed Apr. 14, 2015, the specification ofwhich is hereby incorporated by reference in their entireties.

BACKGROUND (a) Field

The subject matter disclosed generally relates to compounds resultingfrom the complexation of quaternary ammonium compounds with boric acidand/or its derivatives, and methods of making the same.

(b) Related Prior Art

In recent years, the shrimp aquaculture industry is rapidly expanded andaccording to the Food and Agriculture Organization of the United Nations(FAO), approximately 3.5 million metric tons (corresponding to anestimated value $15 billion) by year were produced. Despite the economicimportance, the global shrimp farming industry continues to be plaguedby various diseases which stand out as serious impediments in itsprogress. It is estimated that about of 60% of losses in shrimpaquaculture have been caused by viral pathogens and 20% by bacterialpathogens (Flegel, T. W., Lightner, D. V., Lo, C. F., Owens, L 2008, In:«Diseases in Asian Aquaculture VI». Bonded-Reantaso, M. G., Mohan, C.V., Crumlish, M. and Subasinghe, R. P. Eds, Fish Health Section», AsianFisheries Society, Manila, Philippines. p. 355-378).

Depending on the species of shrimp involved, the clinical manifestationsare different for each diseases, for example pathogens such as TauraSyndrome Virus (TSV) cause cuticular melanized spots. Yellow head Virus(YHV) for yellowing of the cephalothorax and bleaching of the body,particularly White Spot Syndrome Virus (WSSV), as mentioned its name,induced white spots on the inside surface of the carapace, appendagesand cuticle. It is of interest to mention that WSSV is the most seriousand devastating pathogen of farmed shrimp worldwide because it is highlylethal and contagious, killing shrimp quickly. Outbreaks of WSSV diseasehave wiped out within a few days the entire populations of many shrimpfarms throughout the world. Spreading from Taiwan to Asia, then toCentral, South and North America (Zuidema, D., Van Hulten, M. C. W.,Marks, H. Witteveldt, J., Vlak, J. M. 2004. In: Current trends in thestudy of bacterial and viral fish and shrimp diseases, Leung, K. Y.Ed.), World Scientific, Singapore, p. 237-255), the shrimp WSSV iscurrently the only member of both the Whispovirus genus and theNimaviridae family which can infect more than 90 species of aquaticcrustaceans.

In addition to infections caused by virus, many pathogenic bacteria canalso cause serious problems to the farming industries. The mostimportant is due to Vibrio responsible to vibriosis. Generally, thesebacteria are considered to be opportunistic pathogens causing diseasewhen shrimp are stressed. Although the exoskeleton provides an effectivephysical barrier to certain pathogens, Vibrio spp. are among thechitinoclastic bacteria associated with shell disease and may enterthrough wounds in the exoskeleton or pores of crustaceans. Vibriosis isa common problem world-wide, not only responsible for chronicmortalities of crustaceans, but also serious problems for shellfish,flatfish and finfish cultures. It is worth mentioning that problemscaused by vibriosis are common, but are considered minor compared toviral epidemics.

White Spot Syndrome Virus (WSSV)

In 1993, WSSV was first described as white spot disease outbreaks inprawn Penaeus japonicus farmed in Japan. Around the same time, similardisease and mortalities in other prawn species was observed in Taiwanand China, from where it is suspected to have originated. The virus wasknown under various names which are mainly related to baculovirus suchas. «Chinese baculovirus», «Systemic ectodermal and mesodermalbaculovirus» and «White spot baculovirus», etc. Currently, based on itsunique morphological and genetic features similarities, the viruses weregrouped collectively into the white spot virus complex with WSSV beingadopted as the generic virus name. WSSV are now considered by theInternational Committee on Taxonomy of Viruses to represent a new virusgenus, called «Whispovirus», within the family Nimaviridae.

Morphology and Ultrastructure of WSSV

To date, the morphology and ultrastructure of WSSV is not yet fullyunderstood. However, it has been observed that the WSSV virions show anovoid particle morphology with average size about of 300 nm in lengthand 120 nm in diameter. The viral envelope has the structure of anapparently lipidic bilayer membrane surrounded the nucleocapsid that itis tightly packed within the virion.

The WSSV viral envelope consists of at least 35 different proteins, ofwhich VP28 and VP26 are the most abundant, accounting for approximately60% of the envelope. VP28, encoded by open reading frame (ORF) 421(wsv421), is the major envelope protein and several studies suggest thatVP28 may play a crucial role in the initial steps of systemic WSSVinfection in shrimp, particularly as an attachment protein, binding thevirus to shrimp cells, and helping it to enter into the cytoplasm.

With regard to VP26, the product encoded by wsv311 gene, was firstidentified as being associated to the nucleocapsid. It is likely thatthe VP26 is capable of binding to actin or actin-associated proteins.After internalization into the host cell, viruses must be transportednear the site of transcription and replication, where its genome isdelivered. Thus, it has been suggested that as a major component of theviral nucleocapsid. VP26 may help WSSV to move toward the nucleus byinteracting with actin or cellular actin-binding proteins.

The viral genome is a double-stranded circular DNA molecule and the fulllength sequence was submitted to GenBank (Accession number: AF440570).Generally, the genome of WSSV estimated approximately to be 300 kbp andcontains 292967 nucleotides encompassing 184 major ORF. However, only 6%of the WSSV ORFs could be demonstrated a putative function involved innucleotide metabolism, DNA replication, and protein modification (VanHulten, M. C., Witteveldt, J., Peters, S., Kloosterboer, N., Tarchini,R., Fiers, M., Sandbrink, H., Lankhorst, R. K., Vlak, J. M. 2001. 286,7-22).

Strategies for the Control of WSSV

Due to the impact that WSSV has caused to shrimp cultures all over theworld, several approaches have been used for the management of thedisease. However, it is worth noting that at present there is notreatment available to interfere with the unrestrained occurrence andspread of the disease.

Shrimp Anti-WSSV Immune Response

In mammals, active immunity has been practiced for the control of viralinfection symptoms. Active immunity is resulting by the self-immunecapacity stimulated the production of antibodies against pathogens whichare administered under inactivated or attenuated forms into species suchas humans or animal.

In contrast, invertebrates such as crustaceans lack a true adaptiveimmune system and no specific immune function uses antibodies torecognize and destroy non-self material. For this reason, the passiveimmunity is also envisaged and the processes is generated byadministering pathogens to domestic animals such as birds to obtain thecorresponding antibodies, which are then used for the control of shrimpinfection symptoms. However, the in vivo defense mechanism ofinvertebrates is significantly different from the immune mechanism ofvertebrates, and there has been no concrete disclosure about theeffectiveness of passive immunity for the control of infection symptomsof invertebrates such as shrimp.

It is suggested that hemocytes plays an important part in the defensesystem employed by crustaceans against pathogens, since they initiatecoagulation, delay WSSV infection and inhibiting viral replication.However, the precise mechanism of action of hemocyanin is not clear.

Regardless the active or passive immunity, these processes are onlyspecific for one type of pathogen. In this case, it is preferable to usea product with a larger spectrum of action to effectively control at thesame time different pathogens (bacteria and virus).

Treatment of Infected Animals

Even though there are several methods and products developed recently toattempt to control these pathogens, none have been successful and theresearch for new effective products seems urgent and necessary. Untilnow, no commercial reagents with proven abilities to clear completelyWSSV infections or for prophylaxis in the event of outbreaks of WSDexist. Similar observation can be made for other virus such as TauraSyndrome Virus or Yellow head Virus.

Vibrio parahaemolyticus

Many pathogenic bacteria can also cause serious problems to the farmingindustries. The most important is due to Vibrio (gram-negative bacteriain the family Vibrionaceae) responsible to vibriosis. This disease hasbeen reported in penaeid shrimp culture including at least 14 species:Vibrio herveyi, V. splendidus, V. parahaemolyticus, V. alginolyticus, V.anguillerum, V. vulnificus; V. campbelli, V. lischeri, V. damsella, V.pelagicus, V. orientalis, V. ordalii, V. mediterrani, V. logei, etc.

As a control measure antibiotics are generally used against bacterialinfection symptoms including vibrio disease. Recently, due to a largedissemination of antibiotics, the appearance of resistant bacteria hasbecome problematic, and in addition, the administration of antibioticsdoes not guarantee a sufficient control effect. Also, there has not beenany efficacious medicine developed for viral infection symptoms, andaccordingly, it can be said that there is no effective control measure.

Other Pathogens

There, are numerous pathogens responsible for aquatic animal diseases,and they stern from various etiologies such as viruses, bacteria, fungior parasites. It is worth noting that all these pathogens constituteserious problems for aquaculture farming including:

-   1. Taura Syndrome Virus (TSV) is an emerging disease, caused by a    virus in the family Dicistroviridae, genus Cripavirus that affects    Pacific white shrimp in their post-larval, juvenile and sub-adult    life stages. The mortality rate for these life stages can reach up    to 90%;-   2. Yellow Head Virus (YHV) is another emerging disease that affects    Giant Tiger shrimp (Penaeus monodon), especially in the early and    late juvenile life stages, which is highly lethal and contagious,    killing shrimp quickly. YHV belongs to the family Roniviridae;-   3. Other virus such as Infectious Hypodermal and Haematopoietic    Necrosis (IHHNV), Spherical Baculovirus, Spawner-isolated Mortality    Virus Disease, Spring Viremia of Carp (SVC caused by Rhabdoviruses),    Koi Herpes Virus (KHV), Large Mouth Bass Virus (LMBV), Baculovirus    penaei (BP), etc, are also problematic.

Human Pathogens

There numerous bacteria such as: i) Enterobacteriacae (i.e. Escheriacoli, Salmonella, Shigella, etc.); ii) Clostridium botulinum; iii)Listeria monocytogenes, etc. and viral such as Herpesviridae,Retroviridae, Filoviridae (Ebola virus), etc. responsible for serious,even fatal, illness in human.

Though several products have been developed to prevent and treatpathogens in, such as genetic vaccines, these have proven ineffective incommercial operations, impractical to apply for lack of effectivedelivery mechanisms, or expensive.

Therefore, there is a need for novel antipathogenic agents andcompositions comprising the same.

Also, there is a need for novel composition for the treatment orprevention of pathogenic infections.

SUMMARY

In the present invention, the use of boric acid and its derivatives, ina complex with a quaternary ammonium compound, preferably cholinechloride (due to its natural origin such as from phosphatidylcholine),form a quaternary complex able to control infections caused by manybacteria and virus for human and animal, particularly for use in aquaticanimal farming industries.

According to an embodiment, there is provided a compound of formula I,or a pharmaceutically acceptable salts thereof, and stereoisomersthereof:

wherein

R¹, R², R³, and R⁴ are independently selected from alkyl, cycloalkyl, oraryl, optionally substituted with at one or more —OH, and

-   each of said R¹, R², R³, and R⁴ may be optionally connected to    another of said R¹, R², R³, and R⁴;-   R⁵ is selected from —BO₂, —BO₃, —BO₄, —B₂O₃, —B₂O₄, —B₃O₅, —B₃O₇,    —B₄O₇, —B₄O₉—B₅O₆, —O—BR⁶R⁷;-   R⁶ is selected from —H, —OH, alkyl, alkenyl, aryl, —O—R⁸;-   R⁷ is absent or selected from —H, —OH, alkyl, alkenyl, aryl, and    —O—R⁸;-   R⁸ is selected from alkyl, alkenyl, aryl.

The R¹ may be CH₂—CH₂.

The R², R³, and R⁴ may be independently CH₃, CH₂—CH₃, or CH₂—CH₂—CH₃.

The R¹ may be CH₂—CH₂, R², R³, and R⁴ may be independently CH₃.

The R⁵ may be —B₅O₈.

The R⁵ may be —O—BR⁶R⁷.

The R⁵ may be —O—B(OH)₂.

The compound may be selected from the following compounds:

or a combination thereof.

The compound may be a combination of the following compounds:

According to another embodiment, there is provided a pharmaceuticalcomposition comprising a compound of the present invention, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

According to another embodiment, there is provided a compositioncomprising a compound of the present invention, or a pharmaceuticallyacceptable salt thereof, and an acceptable carrier.

According to another embodiment, there is provided a use of a compoundof the present invention or a pharmaceutically acceptable salt thereoffor the manufacture of a medicament for treatment of a pathogenicinfection in a subject.

According to another embodiment, there is provided a use of a compoundof the present invention, or a pharmaceutically acceptable salt hereof,or the composition of the present invention, for treatment of apathogenic infection in a subject.

The subject may be selected from the group consisting of a mammal, afish, a bird, and a crustacean.

The mammal may be selected from the group consisting of a human, abovine, an equine, and an ungulate.

The fish may be selected from the group consisting of a hagfish, alamprey, a cartilaginous fish and a bony fish.

The bird may be selected from the group consisting of chicken, a turkey,and a fowl.

The crustacean may be selected from the group consisting of a shrimp, acrab, a lobster, a langouste.

According to another embodiment, there is provided a method of treatingor preventing a pathogenic infection in a subject in need thereofcomprising:

-   -   administering a therapeutically effective amount a compound of        the present invention, or a pharmaceutically acceptable salt        thereof, or the composition of the present invention, to said        subject.

The subject may be selected from the group consisting of a mammal, afish, a bird, and a crustacean.

The mammal may be selected from the group consisting of a human, abovine, an equine, and an ungulate.

The fish may be selected from the group consisting of a hagfish, alamprey, a cartilaginous fish and a bony fish.

The bird may be selected from the group consisting of chicken, a turkey,and a fowl.

The crustacean may be selected from the group consisting of a shrimp, acrab, a lobster, and a langouste.

According to another embodiment, there is provided a method of treatingor preventing a pathogenic infection in a crustacean in need thereofcomprising:

-   -   administering a therapeutically effective amount a compound of        the present invention, or a pharmaceutically acceptable salt        thereof, or the composition of the present invention, to said        crustacean.

The crustacean may be selected from the group consisting of a shrimp, acrab, a lobster, and a langouste.

The pathogenic infection may be caused by a virus, a microorganism, orcombinations thereof.

The virus may be one of more of White Spot Syndrome Virus (WSSV). TauraSyndrome Virus (TSV), Yellow Head Virus (YHV), Infectious Hypodermal andHaematopoietic Necrosis (IHHNV), Spherical Baculovirus, Spawner-isolatedMortality Virus Disease, Spring Viremia of Carp (SVC caused byRhabdoviruses), Koi Herpes Virus (KHV), Large Mouth Bass Virus (LMBV),and Baculovirus penaei (BP).

The microorganism may be one or more of Vibrio parahaemolyticus, Vibrioharveyi, V. splendidus, V. parahaemolyticus, V. atginolyticus, V.anguillarum, V. vulnificus, V. campbelli, V. fischeri, V. damsella, V.pelagicus, V, orientalis, V. ordalii, V. mediterrani, V. logei, anEnterobacteriacae, Clostridium botulinum, Listeria monocytogenes.

The Enterobacteriacae may be one or more of an Escheria coli, aSalmonella, a Shigella.

Administering may be by feeding said compound, or said pharmaceuticallyacceptable salt thereof, or said composition to said crustacean.

The composition may be a dietary composition.

The composition may be for use in the treatment or prevention of apathogenic infection in a crustacean in need thereof.

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed and claimed is capable ofmodifications in various respects, all without departing from the scopeof the claims. Accordingly, the drawings and the description are to beregarded as illustrative in nature, and not as restrictive and the fullscope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present disclosure will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1: illustrates the synthesis of choline/boric acid complex.

FIG. 2: illustrates the preparation of choline/pentaborate complexobtained by different synthesis methods.

FIG. 3: illustrates FTIR spectra of choline chloride, boric acid,pentaborate and choline complexed with borate and pentaborate obtainedby different synthesis methods.

FIG. 4: Percentage of viability (%) of shrimps L. vannamei treated withfeed containing choline/pentaborate complex (5 mg/g) after infectionwith Vibrio parahaemolyticus.

FIG 5: Percentage of viability (%) of shrimps L. vannamei treated withfeed with choline/pentaborate complex (5 mg/g) after infection withwhite spot syndrome virus (WSSV) homogenates.

FIG. 6: Mean weight increase of shrimp over time fed a commercial dietand a commercial diet supplemented with choline/pentaborate complex, (●)Shrimp group fed with a commercial feed (control group); (Δ) Shrimpgroup fed with the commercial feed but supplemented with 5 mg of thecholine/pentaborate complex/g. Both shrimp groups are fed daily adlibitum for to 28 days.

FIG. 7: Microarray functional annotation of 2 Up-regulated genes frommicroarray of the biological processes. The score is calculated forevery node in the graph and sum of the distances to the GO originalterms.

FIG. 8: Functional Annotation of 2 Up-regulated genes from microarray ofthe molecular function. The score is calculated for every node in thegraph and sum of the distances to the GO original terms.

FIG. 9: Fischer Exact Test 2 Up-regulated GO terms versus all genes ofGO Terms from microarray using α=0.01

FIG. 10: Functional Annotation of 2 Down-regulated genes from microarrayof the biological processes. The score is calculated for every node inthe graph and sum of the distances to the GO original terms.

FIG. 11: Functional Annotation of 2 Down-regulated genes from microarrayof the molecular function. The score is calculated for every node in thegraph and sum of the distances to the GO original terms.

FIG. 12: Fischer Exact Test 2 Down-regulated GO terms versus all genesof GO Terms from microarray using α=0.01.

FIG. 13: RT q-PCR analysis of selected genes related to immune anddigestive proteins of the shrimp, used to validate the DNA microarrayassays.

FIG. 14: illustrate the synthesis of complexes of other quaternaryammonium compounds with boric acid.

FIG. 15: illustrate the synthesis of choline/phenyl boronate complex.

FIG. 16: illustrate the synthesis of choline/myristyl boronate complex.

DEFINITIONS

“Alkyl”, as well as other groups having the prefix “alk”, such as alkoxyand alkanoyl, means carbon chains which may be linear or branched, andcombinations thereof, unless the carbon chain is defined otherwise.Examples of alkyl groups include methyl, ethyl, propyl, isopropyl,butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and thelike. Where the specified number of carbon atoms permits, e.g., fromC3-10, the term alkyl also includes cycloalkyl groups, and combinationsof linear or branched alkyl chains combined with cycloalkyl structures.When no number of carbon atoms is specified, C1-6 is intended.

“Aryl” means a mono- or polycyclic aromatic ring system containingcarbon ring atoms. The preferred aryls are monocyclic or bicyclic 6-10membered aromatic ring systems. Phenyl and naphthyl are preferred aryls.The most preferred aryl is phenyl.

The term “cycloalkyl” is a subset of alkyl and means a saturatedcarbocyclic ring having a specified number of carbon atoms. Examples ofcycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, and the like. A cycloalkyl group generally ismonocyclic unless stated otherwise. Cycloalkyl groups are saturatedunless otherwise defined Cycloalkanes consist of only carbon (C) andhydrogen (H) atoms and are saturated because there are no multiple C—Cbonds to hydrogenate (add more hydrogen to). A general chemical formulafor cycloalkanes would be C_(n)H_(2(n+1−g)) where n=number of C atomsand g=number of rings in the molecule. For those cycloalkanes that haveone ring in their molecules, cycloalkanes can be treated as isomers oftheir alkene counterparts, for example, cyclopropane and propene bothhave the chemical formula C₃H₆. Cycloalkanes with a single ring arenamed analogously to their normal alkane counterpart of the same carboncount: cyclopropane, cyclobutane, cyclopentane, cyclohexane, etc. Thelarger cycloalkanes, with greater than 20 carbon atoms are typicallycalled cycloparaffins.

The term «composition» as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. Such term inrelation to pharmaceutical composition is intended to encompass aproduct comprising the active ingredient(s) and the inert ingredient(s)that make up the carrier, as well as any product which results, directlyor indirectly, from combination, complexation or aggregation of any twoor more of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions or interactions of one ormore of the ingredients. Accordingly, the pharmaceutical compositions ofthe present invention encompass any composition made by admixing acompound of the present invention and a pharmaceutically acceptablecarrier. By «pharmaceutically acceptable» or «acceptable» it is meantthe carrier, diluent or excipient must be compatible with the otheringredients of the formulation and not deleterious to the recipientthereof.

The terms “administration of”, “administer” and/or “administering a”compound should be understood to mean providing, to dispense, to meteout, give to, a compound of the invention of the invention to theindividual or subject in need of treatment by any suitable means, suchas oral administration, parenteral, rectal, transdermal, etc. Accordingto an embodiment for administration to crustacean, the compounds andcomposition of the present invention may be provided through the foodadministered to the subject.

Compounds of Formula I may contain one or more asymmetric centers andcan thus occur as racemates and racemic mixtures, single enantiomers,diastereomeric mixtures and individual diastereomers. The presentinvention is meant to comprehend all such isomeric forms of thecompounds of Formula I.

Compounds of Formula I may be separated into their individualdiastereoisomers by, for example, fractional crystallization from asuitable solvent, for example methanol or ethyl acetate or a mixturethereof, or via chiral chromatography using an optically activestationary phase. Absolute stereochemistry may be determined by X-raycrystallography of crystalline products or crystalline intermediateswhich are derivatized, if necessary, with a reagent containing anasymmetric center of known absolute configuration.

Alternatively, any stereoisomer of a compound of the general structuralFormula I may be obtained by stereospecific synthesis using opticallypure starting materials or reagents of known absolute configuration.

If desired, racemic mixtures of the compounds may be separated so thatthe individual enantiomers are isolated. The separation can be carriedout by methods well known in the art, such as the coupling of a racemicmixture of compounds to an enantiomerically pure compound to form adiastereomeric mixture, followed by separation of the individualdiastereomers by standard methods, such as fractional crystallization orchromatography. The coupling reaction is often the formation of saltsusing an enantiomerically pure acid or base. The diasteromericderivatives may then be converted to the pure enantiomers by cleavage ofthe added chiral residue. The racemic mixture of the compounds can alsobe separated directly by chromatographic methods utilizing chiralstationary phases, which methods are well known in the art.

DETAILED DESCRIPTION

In embodiments, there are disclosed compounds formed by the complexationof boric acid and its derivatives and quaternary ammonium compounds.

Boric Acid and its Derivatives

Boric acid end its salts were registered in 1983 for control ofcockroaches, ants, grain weevils and several beetles. They were alsoused as herbicide, fungicide and wood preservative, even as an insectrepellent in insulation. As an insecticide, boric acid acts as a stomachpoison for ants, cockroaches, silverfish and termites, and as abrasiveto the insects exoskeleton. As an herbicide, boric acid causesdesiccation or interrupts photosynthesis in plants.

Boron is a naturally-occurring element in the earth's crust andbackground levels even circulate in the human bloodstream. According toUnited States Environmental Protection Agency (US EPA. 1993. Prevention,Pesticides, and Toxic Substances. EPA-738-F-93-006), boric acid and itssalts will not pose unreasonable risks or adverse effects to humans orthe environment. Available studies indicate that (ethnical boric acid ispractically nontoxic to birds, fish and aquatic invertebrates, andrelatively nontoxic to beneficial insects. Moreover, the amount of boricacid and its salts used as pesticides are relatively small andsignificant lower than amounts of boron presented naturally in soil andwater.

Borates that may be used in the present invention include but are notlimited to orthoborate, metaborate, triborate, tetraborate, pentaborate,etc. or combination thereof. Also included are boric acid derivativessuch as boronic acids (alkyl- (e.g. myristyl, palmityl, stearyl),alkenyl- or aryl-substituted boric acid such as phenyl boronic acid, 4pyridine boronic acid, etc,) or organoborates (alkyl, alkenyl or arylester borate such as phenyl ester boric acid) or combination thereof.

Quaternary Ammonium Compound and Choline

Quaternary ammonium compounds (cations), also known as quats, arepositively charged polyatomic ions of the structure NR₄ ⁺, R being analkyl group or an aryl group. Unlike the ammonium ion (NH₄ ⁺) and theprimary, secondary, or tertiary ammonium cations, the quaternaryammonium cations are permanently charged, independent of the pH of theirsolution. Quaternary ammonium salts or quaternary ammonium compounds(called quaternary amines in oilfield parlance) are salts of quaternaryammonium cations with an anion.

According to an embodiment, the quaternary ammonium compound is cholineand derivatives thereof, which is a water-soluble nutrient, usuallygrouped within the B-complex vitamins, that plays key roles in manybiological processes. Choline generally refers to the various quaternaryammonium salts containing the N,N,N-trimethylethanol ammonium cation.The cation appears in the head groups of phosphatidylcholine andsphingomyelin, two classes of phospholipid that are abundant in cellmembranes. Choline is the precursor molecule for the neurotransmitteracetylcholine, which is involved in many functions including memory andmuscle control. Choline must be consumed through the diet for the bodyto remain healthy. It is used in the synthesis of the constructionalcomponents in the body cell membranes. Despite the perceived benefits ofcholine, dietary recommendations have discouraged people from eatingcertain high-choline foods, such as egg and fatty meats. The 2005National Health and Nutrition Examination Survey stated that only 2% ofpostmenopausal women consume the recommended intake for choline.

According to another embodiment, quaternary ammonium compound ispreferably, but not limited to, choline chloride (2-hydroxyethyltrimethyl ammonium chloride) or its derivatives possessing on the alkylchain at least a free hydroxyl group, i.e. (2-Hydroxyethyl)triethylammonium chloride, (2-hydroxypropyl)trimethyl ammonium chloride;(2,3-dihydroxypropyl)trimethyl ammonium chloride, etc.

Therefore, in embodiments, there is disclosed an antibacterial andantiviral compound based on borate or borate derivatives which arecomplexed with a quaternary ammonium.

In embodiments, there is disclosed a compound of formula I, or apharmaceutically acceptable salts thereof, and stereoisomers thereof:

wherein

R¹, R², R³, and R⁴ are independently selected from alkyl, cycloalkyl, oraryl, optionally substituted with one or more —OH, and

each of the R¹, R², R³, and R⁴ may be optionally connected to

another of the R¹, R², R³, and R⁴;

R⁵ is selected from —BO₂, —BO₃, —BO₄, —B₂O₃, —B₂O₄, —B₃O₅, —B₃O₇, —B₄O₇,—B₄O₉—B₅O₈, —O—BR⁶R⁷, —R⁹—BR⁶R⁷;

R⁶ is selected from —H, —OH, alkyl, alkenyl, aryl, —O—R⁸;

R⁷ is absent or selected from —H, —OH, alkyl, alkenyl, aryl, and —O—R⁸;

R⁸ is selected from alkyl, alkenyl, aryl;

R⁹ is selected from alkyl, alkenyl, and aryl.

According to an embodiment, the R¹ may be C₁₋₆, alkyl, linear orbranched. According to an embodiment, the R¹ may be CH₂—CH₂.

According to another embodiment, the R², R³, and R⁴ may be C₁₋₃ alkyl.According to another embodiment, the R², R³, and R⁴ may be independentlyCH₃, CH₂—CH₃, or CH₂—CH₂—CH₃.

According to another embodiment, the R⁵ is —B₅O₈.

Indeed, according to an embodiment, boric acid or its derivativescomplexed with choline are preferably used in the present invention, dueto several advantages such as naturally occurring; low toxicity;inexpensive and easy to manufacture.

According to another embodiment, the compound of formula I, or apharmaceutically acceptable salts thereof, and stereoisomers thereof.may be selected from the following compounds:

and combinations thereof.

The invention includes the compounds as shown, and also includes (wherepossible) individual diastereomers, enantiomers, and epimers of thecompounds, and mixtures of diastereomers and/or enantiomers thereofincluding racemic mixtures. Although the specific stereochemistriesdisclosed herein are preferred, other stereoisomers, includingdiastereomers, enantiomers, epimers, and mixtures of these may also beuseful in treating pathogenic infections. Inactive or less activediastereoisomers and enantiomers are useful for scientific studiesrelating to the target pathogens and the mechanisms of action of thecompounds of the present invention.

The compounds disclosed herein may be used in pharmaceuticalcompositions comprising (a) the compound(s) or pharmaceuticallyacceptable salts thereof, and (b) a pharmaceutically acceptable carrier.The compounds may be used in pharmaceutical compositions that includeone or more other active pharmaceutical ingredients. The compounds mayalso be used in pharmaceutical compositions in which the compound ofFormula I or a pharmaceutically acceptable salt thereof is the onlyactive ingredient.

The compounds disclosed herein may be used in compositions comprising(a) the compound(s) or acceptable salts thereof, and (b) an acceptablecarrier. The compounds may be used in compositions that include one ormore other active ingredients. The compounds may also be used incompositions in which the compound of Formula I or an acceptable saltthereof is the only active ingredient.

According to another embodiment, there is disclosed a use of a compoundof the present invention, or a pharmaceutically acceptable salt thereoffor the manufacture of a medicament for treatment of a pathogenicinfection in a subject.

Also disclosed is the use of a compound of the present invention, or apharmaceutically acceptable salt thereof, or the composition of thepresent invention, for treatment of a pathogenic infection in a subject.

According to another embodiment, there is disclosed a method of treatingor preventing a pathogenic infection in a subject in need thereofcomprising administering a therapeutically effective amount a compoundof the present invention, or a pharmaceutically acceptable salt thereof,or the composition of the present invention, to the subject.

According to an embodiment, the pathogenic infection may be a bacterialinfection, a viral infection, a fungal infection, a parasite infection,or a combination thereof. Examples of bacterial infections include butare not limited to Vibrionaceae infection, a Enterobacteriacaeinfection, a Clostridium botulinum infection, a Listeria monocytogenesinfection. Examples of viral infection include but are not limited to aTaura Syndrome Virus (TSV) infection, a Yellow Head Virus (YHV)infection, a Infectious Hypodermal and Haematopoietic Necrosis (IHHNV)virus infection, a Spherical Baculovirus infection, a Spawner-isolatedMortality Virus Disease infection, a Rhabdovirus infection, a Koi HerpesVirus (KHV) infection, a Large Mouth Bass Virus (LMBV) infection, aBaculovirus penaei (BP) infection, Herpesviridae infection, aRetroviridae infection, a Filoviridae infection, and an HIV infection.

According to an embodiment, the subject may be a mammal, a fish, a bird,and a crustacean. The mammal may be a human, a bovine, an equine, anungulate, etc. The fish may be a hagfish, a lamprey, a cartilaginousfish and a bony fish. Said bird may be a chicken, a turkey, a fowl. Saidcrustacean may be a shrimp, a crab, a lobster, a langouste, etc.

According to another embodiment, there is disclosed a method of treatingor preventing a pathogenic infection in a crustacean in need thereofcomprising administering a therapeutically effective amount a compoundof any one of the present invention, or a pharmaceutically acceptablesalt thereof, or the composition of the present invention, to thecrustacean.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

These examples further illustrate the method of production of boratederivatives and method to complex with quaternary ammonium compound,preferably choline (2-hydroxyethyl)trimethyl ammonium chloride (FIG. 1).

EXAMPLE 1 Preparation of Choline/Borate Complex

The choline/borate complex (FIG. 1) is essentially prepared in saturatedaqueous solution in order to facilitate obtaining the final product bycrystallization at low temperature. The concentrations of choline andboric acid are preferably used in an equimolar ratio (1:1).

1-1—Complexation of Choline Chloride with Boric Acid

An amount of 123.66 g of boric acid is dispersed in 400 mL of distilledwater at 90° C. The solution is under strong stirring. After dispersionof Boric acid, an amount of 279.24 g of choline chloride is slowly addedin the boric acid solution and the temperature is maintained at 60° C.When choline chloride is added, the solution is cloudy, but become clearafter 1 h heating, always under strong stirring (˜600 rpm). The reactionis continued during at least 2 h.

1-2—Crystallization of the Choline/Borate Complex

At the end of the reaction, the temperature is gradually reduced inorder to cool down slowly the solution, while gentle stirring (˜200rpm). When the temperature reached between 35-40° C., the solution istransferred in a plastic container and the stirring is reduced at 150rpm to promote crystal formation. At this moment, some ice can be addedinto the water bath to accelerate the cooling-down processing. Once thetemperature is about of 15° C., the solution is refrigerated between4-6° C. for 24 hours. The crystals are then collected by decantation orby filtration using a 24-cm Whatman filter paper No 1 under a negativepressure (vacuum) about of 40 kPa. The collected crystals are dried inan oven at a temperature ranging from about 35-40° C. for at least 24 h.

EXAMPLE 2 Preparation of Choline/Tetraborate Complex

The complexation of choline and tetraboric acid is prepared underidentical conditions as described previously in EXAMPLE 1, except thatdisodium tetraborate decahydrate is used instead of boric acid.

EXAMPLE 3 Preparation of Choline/Pentaborate Complex

The synthesis of choline/pentaborate complex could be prepared in twoways. Firstly, one approach consists in preparing first choline/boratecomplex which reacts with disodium tetraborate decahydrate to formcholine/pentaborate complex. Alternatively, another approach consists inprimarily synthesizing sodium pentaborate followed the complexing withcholine.

3.1—Method I:

3-1-1—Preparation of Choline/Borate Complex

The complexation of choline and boric acid is prepared under identicalconditions as described previously in EXAMPLE 1 (FIG. 1).

3-1-2—Complexation of Choline/Borate with Disodium TetraborateDecahydrate

When the reaction of complexation choline/boric acid is achieved, thetemperature of the solution is reduced to about 60° C. Then, an amountof 127.39 g of disodium tetraborate decahydrate is added in thesolution, under strong stirring in order to react with choline/boricacid previously obtained (FIG. 2, Method 1). After complete dispersion,the reaction is continued at least 2 h and the temperature of thesolution is gradually reduced to room temperature and gentle stirring(150-200 rpm) to favor the crystallization. When the temperature hasreached at room temperature, the solution is refrigerated for 24 h andcholine/pentaborate crystals are collected by filtration and dried for24 h at 40° C.

3-2—Method II

3-2-1—Preparation of Sodium Pentaborate

A volume of 800 mL of distilled water is introduced in a glass temperingbeaker and heated at the temperature of about 60° C. When the settemperature is reached, an amount of 350 g of disodium tetraboratedecahydrate and of 340 g of boric acid are introduced in the beakeralternately, portion by portion (approximately 20 g for each), undergentle stirring. Dissolution of all chemicals is ensured before addingthe next portion, without stopping stirring.

Once all chemicals are completely added, the temperature of the solutionis maintained at 60° C., always under stirring for at least 30 min. Toobtain sodium pentaborate powders, the solution is slowly cooled downwith moderate stirring (about of 200 rpm). When the temperature reaches25° C., the solution is refrigerated at 4° C. during 24 h. Theprecipitate is collected by filtration in a plastic container and storedat 40° C. at least 24 h before use.

3-2-2—Complexation of Pentaborate with Choline

An amount of 300 g of sodium pentaborate crystals (previously obtained)is introduced in a glass tempering beaker containing 350 mL distilledwater, under) gentle stirring at a temperature 55±2° C. After completelydissolving, an amount of 140 g of choline chloride is slowly added inthe pentaborate solution, always under stirring and the reaction isstarted for at least 1 h (FIG. 2, Method 2).

The choline/pentaborate complex crystals are obtained by refrigerationunder identical conditions as described previously for preparation ofsodium pentaborate. Indeed, the choline/pentaborate solution is slowlycooled down with moderate stirring (about of 200 rpm). When thetemperature reaches 25° C., the solution is refrigerated at 4° C. during24 h. The precipitate is collected by filtration in a plastic containerand stored at 40° C. at least 24 h before use. The predicted structureof choline/pentaborate complex is presented in FIG. 2.

EXAMPLE 4 Characterization of Complex of Choline/Borate and itsDerivatives

4-1—Determination of Quaternary Ammonium Compound in the Complex

4-1-1—Spectrophotometric Assay Based on Dragendorff Reagent

In order to estimate the quaternary ammonium compounds (QAC) of cholinederivatives, a spectrophotometric assay based on Dragendorff reagent isused as described by Stumpf (Stumpf, D. K. 1984. Plant. Physiol., 75,273-274) with slight modifications. The principle consists in usingbismuth nitrate and sodium iodide which form a complex and cause theprecipitation of QAC with a development of a brick red color in thereaction medium. Practically, Dragendorff reagent is prepared by mixingequal volumes of bismuth nitrate 0.35 M in acetic acid 20% (v/v) andsodium iodide 2.45 M (in distilled water). Then, an amount of 100 μL ofmixture is added in 10 μL of sample. The standard curve is made byplacing 1.0-8.0 μL of a stock solution of choline (0.500 g/ml) intoplastic 1.5 mL microcentrifuge tubes. Sample and standard solutions arecentrifuged for 3 min at 10,000 g and the supernatants are completelyremoved. The obtained precipitates are then dissolved in 1 mL of 2.45 MNal solution under strong stirring for at least 15 min and diluted byadding 99 mL of 0.49 M Nal. Finally, the samples are recorded byspectrophotometry at 420 nm (maximum absorption) using solution of 0.49M Nal as a blank.

Generally, the quaternary ammonium compounds are detected in all sampleswhich the ratio of choline/borate (tetraborate or pentaborate) complexis 1:1 (equimolar complex). No significant difference is observed forcholine/pentaborate complexes obtained from method 1 and 2, as describedin EXAMPLE 3.

4-1-2—Elemental Analysis

Analysis of the element compositions of choline/borate or itsderivatives complexes allows confirming the structure of the complexformed between choline and different borate or its derivatives. Nosignificant difference is observed between the ratio ofcholine/pentaborate obtained by the method 1 and 2, as described inEXAMPLE 3.

4-1-3—FTIR Analysis

FTIR spectra are recorded on a Spectrum One (Perkin Elmer, Canada)instrument equipped with a Universal Attenuated Total Reflectance (UATR)device. All samples including borate or its derivatives and borate orits derivative complexes are recorded under powder (20 mg) forms in thespectral region (4000-650 cm⁻¹) with 24 scans/min at a 4 cm⁻¹resolution.

FIG. 3 illustrates FTIR spectra of choline chloride, boric acid,pentaborate and choline complexed with borate and pentaborate obtainedby different synthesis methods.

For planar boric acid FTIR spectrum, the stretching vibration of theB—O—H bands are observed at 3220 cm⁻¹. For absorption bands located at1430 and 1195 cm⁻¹, they are respectively assigned for the asymmetricB—O stretching vibration and the in-plane B—O—H bending. With regard forpentaborate, similar observation for the stretching vibration of theB—O—H bands is noticed at 3220 cm⁻¹. However, a new absorption bandappeared at about 3380 cm⁻¹ and is possibly due to H—O—H (free O—H fromwater). Additionally, B—O absorption bands contributed for theasymmetric B—O stretching vibration and the in-plane B—O—H bending areshifted to 1375 and 1140 cm⁻¹. A new band also observed at 1300 cm⁻¹ andcould be due to the B—O—B stretching vibration.

When boric acid or pentaborate are complexed with choline, similar FTIRspectral profiles are observed. In the spectral regions 3500-3000 cm⁻¹,the stretching vibrations of the O—H bands are observed at 3380 and 3220cm⁻¹ which are respectively assigned for the absorption band of H—O—Hand the absorption band of B—O—H. In the spectral region 1500-1000 cm⁻¹,the stretching vibrations for B—O are located at 1375 and 1300 cm⁻¹ andthe in-plan B—O—H bending is noticed at 1140 cm⁻¹. A slight shoulderobserved at 1410 cm⁻¹ seems to contribute to quaternary ammonium CH₃—N⁺(from choline). Also, the absorption bands at 1080 and 1010 cm⁻¹ arerespectively attributed to C—N and C—O stretching vibrations.

EXAMPLE 5 Toxicity Evaluation of Choline/Borate and Choline/BorateDerivative Complexes for Shrimp Litopenaeus vannamei

5-1—Collection and Maintenance of Experimental Animals

Shrimp, Litopenaeus vannamei (post-larvae about 12-16 day-old), areobtained from a commercial hatchery in La Paz (BCS, Mexico) andmaintained in 1000 L fiber glass tanks with air-lift biological filtersat room temperature in water with a salinity of 35 parts per thousand(ppt).

Natural seawater is used in all the experiments. It is obtained from theEnsenada de La Paz after removing the sand and other suspended particlesin sea water. Finally, the seawater is sterilized with UV-lamp beforeuse for the experiments. The tanks are individually aerated through airstones connected to a high-volume air blower. Partial water changes aremade once a week to maintain the water quality. The shrimps areinitially fed Artemia sp and are weaned onto a commercial diet(containing 35% crude protein, Purina Brand) when they reached 20 daysof age (post-larvae 20 day-old). Daily, temperature and pH value arerecorded; salinity is measured with a Salinometer (Aquafauna, Japan) anddissolved oxygen is estimated by the Winkler method (Strickland andParsons, 1972).

5-2—Toxicity Evaluation of Choline/Borate and Choline/Borate DerivativeComplexes

White shrimp (L. vannamei) from intensive culture ponds are selected andacclimated to the experimental conditions during one week beforestarting the trial. Indeed, shrimp are placed in 1000 L circular tankswith filtered sea water at 38 parts per thousand (ppt) and constantaeration, fed ad libitum with a commercial pellet containing 35% crudeproteins.

After the acclimatation period, different concentrations (1, 5 and 10mg/g) of choline/borate or its derivatives are physically mixed withcommercial pellet feeds and fed in the identical conditions (ad libitum)during 15 days. Globally, there are 6 groups are investigated in thisstudy:

-   -   1. Boric acid;    -   2. Tetraborate;    -   3. Pentaborate;    -   4. Choline/borate complex;    -   5. Choline/tetraborate complex;    -   6. Choline/pentaborate complex.

Results show that there is no significant difference between group ofcontrol and groups treated with boric acid or its derivatives complexedwith choline. These results suggest that no toxicity are apparent forwhite shrimp L. vannamei for doses of boric acid or its derivativecomplexes lower than 10 mg/g of shrimp feed.

EXAMPLE-6 Preparation for Challenge Studies 6-1—Preparation forBacterial Assays 6-1-1—Bacterial Strains and Culture Conditions

Virulent bacterial strains Vibrio parahaemolyticus CAIM 170 (Collectionof Aquatic Important Microorganisms, CIAD, Mexico) are used in thisstudy (Roque et al., 1998). These strains are maintained in Tryptone SoyBroth (TSB) containing 2.5% (w/v) NaCl and 15% (v/v) of glycerol at −80°C. Prior to use, a cryovial is thawed and inoculated into 5 mL of TSBwith 2.5% (w/v) NaCl and incubated overnight at. 37° C. in a rotaryshaker (200 rpm) for activation. Thereafter, a volume of 2 mL of theovernight culture is transferred to 100 mL of TSB and reincubated in thesimilar activation conditions (37° C., 200 rpm).

Density of bacteria is measured by spectrophotometry at 600 nm for every30 min. At the same time, an aliquot is withdrawn for viable countdetermination by plating serial dilutions on Tryptone Soy Agar with 1%NaCl. The plates are incubated for 24 h at 37° C. A growth curve wasprepared by plotting the viable count (x-axis) against optical densityat 600 nm values (y-axis). This graph was used to determine the viablecell count during further spiking studies. For the challenge, controlgroups are included in all trials:

-   -   A positive control group of shrimp is treated with pathogen V.        parahaemolyticus;    -   A negative control group of shrimp, instead of receiving the        pathogen, is treated with a sterile saline solution (without        pathogens).

Preliminary trials showed that a V. parahaemolyticus (VP) suspension of1×10⁶ colony-forming units (cru)/mL could kill 60% of the shrimppopulation in 24 h and about of 70% in 96 h, whereas a group treatedwith a non-virulent V. parahaemolyticus strain showed the cumulativeshrimp mortality at 96 h is less than 10%.

6-1-2—Optimization of Real-Time Polymerase Chain Reaction Assay forQuantitative Determination of V. parahaemolyticus

The primers used In this assay are Vp-ToxR q-PCR (Vibrioparahaemolyticus ToxR gene quantitative Polymerase Chain Reaction) 176 F(forward primer, SEQ ID NO:1: GGA AGT TTT AAC CCG TAA CGA GC) and 176 R(reverse primer, SEQ ID NO: 2: GGT ACA AAT GAG TTG ATA GCC TCG) anddesigned as described by Untergasser et al. (2007) using the software«Primer3Plus», with the following parameters:

-   -   Primer length: 18-24 bp;    -   GC content: 35-65%    -   Melting Temperature. (Tm): 58C-60° C.;    -   Product size: 80-250 bp (Wang, X. and Seed, B. 2007. In:        «Real-time PCR». Derek M. Tevfik. Ed. Taylor & Francis. New        York. USA. p. 93-105).

Thermodynamic parameters of each primer are evaluated to checkprimer-dimer and secondary structure using the software «Oligoevaluator»(Sigma-Aldrich™) and «Primer digital» (Kalendar. R., Lee, D., Schulman,A. H. 2011. Genomics, 98, 137-144). These primers yielded a 176 byamplicon and are suspended in nuclease free water to make a workingsolution of 10 pmol/μL.

To establish the standard curve, serial dilutions are done with the DNAextracted and purified from culture of V. parahaemolyticus CALM 170using the following mixture:

-   -   5 μL of SSO-Fast supermix evagreen (BIORAD, USA);    -   1 μL of Vp-ToxR q-PCR 176F (10 pmol/μL);    -   1 μL of Vp-ToxR q-PCR176R (10 pmol/μL);    -   2 μL of DNAse-free water and 1 μL of template as DNA.

Quantitative PCR is performed on a Rotor gene 6000 Real-Time PCR system(Qiagen™). Amplification conditions are carried out as follows:

-   -   initial activation at 50° C. for 2 min;    -   initial denaturation at 95° C. for 10 min and followed by 45        cycles of denaturation at 95° C. for 15 s    -   primer annealing at 60° C. for 20 s and elongation at 72° C. for        30 s and a final elongation at 72° C. for 5 min.    -   A melting curve analysis is performed at the end of the        amplification using the following conditions: 65° C.-80° C. (1°        C./s).

The data are analyzed using the software Rotor Gene-Q Pure Detection(1.7 Build 94).

-   -   6-1-3—Enrichment Media and Samples for Detection of V.        parahaemolyticus

In this study, an enrichment medium is used to evaluate their effect ondetection of V. parahaemolyticus. The enrichment medium used is alkalinepeptone water (APW; 1% peptone, 1% NaCl, pH 8.5; Tyagi et al., 2009).Practically, an amount of 5 g of shrimp is collected and placed on ice.Immediately, these shrimp are homogenized in phosphate buffer at pH 7.5with a Polytron homogenizer, under sterile conditions. A volume of 1 mLof homogenized material is inoculated in APW medium and incubatedovernight at 37° C. for 24 h in a rotary shaker at 200 rpm.

6-1-4—V. parahaemolyticus DNA Extraction from APW Medium

A volume of 1 mL of APW medium is collected in a microcentrifuge tube.The tube containing medium is centrifuged at 10,000 g for 10 min and thepellet is collected and washed with miliQ sterile water and suspended in400 μL in miliQ sterile water. Further centrifugation at 10,000 g for 5min and the tubes are incubated at 98° C. for 20 min before centrifugedat 5,000 g for 5 min. The supernatant is collected in a newmicrocentrifuge tube and stored at −20° C.

6-2—Preparation for White Spot Syndrome Virus (WSSV) Assays

6-2-1—WSSV Stock and In Vivo Titration

The virus is isolated from WSSV-infected adult L. vannamei shrimpobtained from commercial farms located in Sinaloa, Mexico in 2013. Virusstocks were purified from infected shrimp homogenates by improveddifferential centrifugation as described previously by Du et al, (Du, H.H., Fu, L. L., Xu, Y. X., Kil, Z. S., Xu. Z. R. 2007. Aquaculture,262:532-534) and then stored at −80° C. For assays, the WSSV stock isserially diluted to prepare solutions containing various target copynumbers determined by competitive PCR.

A volume of 20 μL of different dilutions of the tissue homogenate(containing WSSV in 330 mM of NaCl) is injected intramuscularly infourth or fifth abdominal segment of L. vannamei using an insulinneedle. The mortality is recorded twice a day and dead shrimp are testedby PCR to highlight the presence of WSSV.

A dilution of WSSV containing 1×10⁶ copies is appropriated to use insubsequent experiments (Du et at. 2006). In the present study, thisdilution containing 1×10⁶ copies is approximately corresponding to 3% ofWSSV suspension which can kill 50% of shrimps in 48 h, and 100% ofshrimps in 96 h; on the other hand, a virus dilution at 1% of WSSVsuspension can kill 80% of the shrimps in 120 h.

6-2-2—Detection of WSSV Using q-PCR Technique

After 48, 72 and 96 h after infection, haemolymph is collected from theventral sinus using a sterile syringe containing 500 μL of anticoagulantsolution (pH 7.3, at 4° C.) as described by Vargas-Albores et al.(Vargas-Albores, F., Guzmán, M. A., Ochoa, J. L. 1993. Comma. Biochem.Physiol., Part A, 106, 299-303):

-   -   450 mM NaCl;    -   10 mM KCl;    -   10 mM HEPES;    -   10 mM EDTA

The collected haemolymph is centrifuged at 12,000 g for 20 min at 4° C.and the precipitate is resuspended in TRIzol reagent™. According to themanufacturer's instructions, total RNA is extracted and the pellets oftotal RNA are resuspended in 15 μL of RNAse free water. Total RNA isquantified using Nanodrop™ 1000 (Thermoscientific) using an amount of 1μg of total RNA treated with DNAse I (Invitrogen™). The cDNA synthesisis performed using «Go Script Reverse Transcription System» (Promega).The sequence of used primer WSV230F is SEQ ID NO:3: GCT GGT GGG GGA TGATAC TA, and that of primer WSV230R is SEQ ID NO:4: GTC TCC CGT CAC CGTCTT TA, as described by Gomez-Anduro et al. (Gomez-Anduro, G. A.,Barillas-Mury, C. V., Peregrino-Uriarte, A. B., Gupta, L, Gollas-Galvam,T., Hernandez-Lopez, J., Yepiz-Plascencia, G. 2006, Develop. Comp.Immunol., 30: 893-900). The q-PCR is performed as follows:

-   -   5 μL of SSO-fast qPCR supermixes evaGreen (BIORAD, USA);    -   1 μL of WSV230F (10 pmol/μL) and 1 μL of WSV230R (1.0 pmol/μL);    -   2 μL of DNAse-free water;    -   1 μL of template as cDNA from hemocytes.

Initial denaturation at 95° C. for 10 min followed by 40 cycles ofdenaturation at 95° C. for 15 s, primer annealing at 58° C. for 20 s,elongation at 72° C. for 30 s and a final elongation at 72° C. for 5min. A melting curve analysis is performed at the end of theamplification using the following conditions: 70° C.-95° C. (1° C./s).The data is analyzed using the software Rotor-Gene-Q Pure Detection (1.7Build 94).

6-3—Challenge Studies

6-3-1—Experimental System

A static experimental system is used in consisting of 24, 20-1 fiberglass tanks. Each tank containing filtered and UV-sterilized seawater(at 35 ppt., pH 8.0) is individually aerated by an air stone at 27° C.

6-3-2—Experimental Procedure

6-3-2-1—Acclamation

An amount of twenty (20) shrimp are introduced to each tank filled with30-L of UV-sterilized seawater. The shrimp are left to adapt to thesystem (acclamation) during 2 days. Shrimp is'fed with pelletizedcommercial feed mixed with different quantities of choline/pentaboratecomplex during 28-days for further challenges with WSSV and a pathogenicVibrio parahaemolyticus strain,

6-3-2-2—Challenge Test

The shrimp are either challenged in triplicate by injection with a knownconcentration of White Spot Syndrome Virus (1-3% viral suspension) or apathogenic Vibrio parahaemolyticus (1×10⁶ cfu/mL) strain. There are twocontrol groups:

-   -   1. Positive control groups—Shrimp that are not previously        exposed with any borate or its derivatives and are either        injected with a known concentration of WSSV or pathogenic VP;    -   2. Negative control groups—Shrimp are not fed with borate or its        derivatives and are not treated with any pathogens; however,        shrimp are injected with a saline solution, instead pathogens.

After the pathogen challenge, treated and control shrimp groupscontinued receiving the corresponding feed. The shrimp mortalitypercentage is recorded daily. Shrimps are observed for 96 to 120 h aftereach challenge and recorded water temperature and mortality. Shrimpsamples are taken on the first day, during challenge and 72 hpost-challenge. All shrimp samples are stored at −80° C. in RNA latterfor further q-PCR analysis of pathogen loadings and expression of immunerelated genes.

6-3-2-3—Determination of Mortality Shrimp

Dead shrimp are recorded daily during 120 hours. All dead shrimp samplesare stored at −80° C. in RNA latter for further q-PCR analysis ofpathogen loading and expression of immune related genes.

6-4—Results of Challenge Studies with Boric Acid or its Derivatives UsedAlone or Complexed with Choline

Shrimp challenged with virulent V. parahaemolyticus (FIG. 4) showedafter 96 h that the shrimp survivals for the negative (not infected withpathogen and not treated with any borate complexes) control group are100%. For positive (pathogen infected, but not treated with any boratecomplexes) control group, the survival rate is about 30%. Similarsurvival rate for borate, tetraborate or pentaborate (not complexed withcholine) are observed at different doses (1, 5 and 10 mg/g of commercialpellets).

In contrast, high survival rates (>60%, p<0.05) are observed forcholine/borate, choline/tetraborate and choline/pentaborate complexes,

Similar observation for shrimp challenged with WSSV (infected with 1% ofWSSV suspension homogenate), the shrimp survival for the negative (notinfected with pathogen and not treated with any borate complexes)control group are 100% after 96 h. For positive (pathogen infected, butnot treated with any borate complexes) control group, the survival rateis about 30%. Similar survival rate for borate, tetraborate orpentaborate (not complexed with choline) are observed at different doses(1, 5 and 10 mg/g of commercial pellets). In contrast, high survivalrates (>60%) are observed for choline/borate, choline/tetraborate andcholine/pentaborate complexes.

It is worth mentioning that for all complexes tested with V.parahaemolyticus, the best survival (>80%) results are obtained withcholine/pentaborate complex supplemented at dose of 5 mg/g of commercialpellet feed. Consequently, the choline/pentaborate complex is selectedfor subsequent studies.

6-4-1—Results of Choline/Pentaborate Complex Challenged with Vibrioparahaemolyticus

For shrimp infected with V. parahaemolyticus (1×10⁶ cfu/mL) and treatedwith choline/pentaborate complex at dose 5 mg/g of feed, the survival(FIG. 4) is 84% (p<0.01) after 96 h, whereas the survival of shrimpinfected with V. parahaemolyticus and not treated withcholine/pentaborate is about 30%.

6-4-1—Results of Choline/Pentaborate Complex Challenged with WSSV

There are no statistical differences between replicates for the sametreatments (p>0.05), as the standard deviation between samples is lowerthan 3%.

Results show that there are statistical differences in shrimp challengedwith a 3% WSSV suspension. Survival after 48 hours for shrimp from thepositive control (infected) is about of 80% and for shrimp fed withcholine/pentaborate complex at dose 5 mg/g is 95% survival(statistically different p<0.05). After 96 h post-challenge, only 10% ofthe positive control shrimp are alive. There is a marginal improvementfor shrimp fed with choline/pentaborate complex at doses the 1 and 5mg/g, where the survivals rates are about of 40%.

In contrast, when shrimp are challenged with 1% WSSV suspension (FIG.5), there is a statistical difference in survival (p<0.05). After 72 h,shrimp from the positive control (infected) had about of 65% survivalwhereas shrimp fed with 5 mg of choline/pentaborate complex/g of feedhad 95% survival. After 96 h post-challenge, about 35% survival forshrimps in the positive control and 85% survival for shrimp fed with 5mg of choline/pentaborate complex by gram of feed (p<0.05).

The use of functional feeds that contain antipathogenic compounds isconsidered fundamental in the strategy to prevent early infectiousdiseases in shrimp, such as Early Mortality Syndrome (EMS) and WhiteSpot Syndrome Virus (WSSV). Further, some antimicrobial compounds, suchas the ones tested in this trial, appear to be able to disrupt bacterialcommunication that activate certain genes associated to the release oftoxins. This quorum quenching represents a significant alternative tothe use of antibiotics.

Data analysis shows that the choline/pentaborate complex formulationsupplemented at a 5 mg/g in feed diets is effective in reducing impactof WSSV infection when following standardized injection protocols. Thisrepresents a major breakthrough for the control of a disease thataffects a significant number of commercial shrimp production operations,from hatcheries to grow out facilities. Though several products havebeen developed to prevent and treat WSSV, such as genetic vaccines,these have proven ineffective in commercial operations, impractical toapply for lack of effective delivery mechanisms, or expensive.Choline/pentaborate complex is not toxic, simple to manufacturing,stable, easy to administer and relatively inexpensive.

The use of choline/pentaborate complex as additive in commercial dietsprovides an efficient mechanism for microbial, activity control in theshrimp gut that may contribute to the solution of the mass mortalitiesassociated to V. parehemolyticus and WSSV in commercial shrimp farms.

EXAMPLE-7 Highlighting of Expression of Immune Related Genes in ShrimpLitopenaeus vannamei by Choline/Pentaborate Complex

7-1—RNA Extraction

RNA is extracted using TRIzol® and the Qiagen RNeasy® Mini Kit (Qiagen,Germantown, Md., USA). Shrimp samples immersed in TRIzol® are placed inthe FastPrep®-24 (MP Biomedicals LLC, Solon, Ohio, USA) and homogenizedduring 4° C. for 40 s pulses at 6 m/s. A volume of 200 μL of chloroformis added to a 1.5 mL RNase free tube containing the homogenate materialand inverted 15 times. Samples are incubated at room temperature for 3min before centrifuged at 8000 g for 15 min at 4° C. using the Eppendorfcentrifuge. The supernatant is carefully transferred into a new 1.5 mLRNase free tube and the extraction continued following themanufacturer's protocol.

7-2—Assessing RNA Quality and Quantity

The quality and quantity of RNA are determined by the NanoDrop™ 1000spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) andBio-Rad™ (Bio-Rad, Hercules, Calif., USA) using denatured gelelectrophoresis with formaldehyde and buffer MOPS 1× (Sambrook, J. 1989.in: «Molecular cloning—a laboratory manual, 2nd ed». J. Sambrook, E. F.Fritsch, T. Maniatis Eds. Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press).

7-3—Microarray and Scanning

Microarrays are designed based on publically available gene sequencesand ESTs determined from cDNA libraries representing multiple tissues ofmale and female L. vannamei shrimp, and/or of various physiologicalconditions.

In total, a length of oligonucleotides about of 61440-mer is included inthis study: 39592 shrimp ESTs; 1216 Quality control spots; 112 positivecontrol Alexa-555 dye; 80 positive control Alexa 647 dye; 20320Unigenes; 16 astringent positive control alexa-555: 16 astringentpositive control Alexa-647 dye and 88 negative control empty aresubmitted to the MYcroarray (University of Michigan, ChemicalEngineering Department) for printing on aminosaline glass slides. Theexposure and reference sample RNAs are adjusted to final volume 19 μLwith oligo d(T) (2 μg/μL) and random primers. (1 μg/μL) in watermolecular biology grade. The samples are incubated at 70° C. for 10 minand are then chilled in ice. The samples are mixed finally with 5×Reaction Buffer Superscript II kit (Invitrogen, Life technologies,Carlsbad Calif., USA): 25 mM MgCl₂; aminoallyl-dNTP with dUTP (3:1); 0.1M DTT and 200 U/μL Superscript II Reverse Transcriptase. The mix isincubated at 25° C. for 10 min and then at 42° C. for 2 hours. RNAhydrolysis is made with 5 μL of NaOH 1 N and 1 μL of EDTA 0.5 M. Themixture is incubated at 65° C. for 10 min, and then added 25 μL of HEPES1 M at pH 7.5. The choline/pentaborate complex exposed samples (n=6) arelabeled with Alexa-647 dye Molecular Probes™ (Life Technologies, EugeneOreg., USA) fluorescent dye and the reference samples (n=6) are labeledwith Alexa-555 dye Molecular Probes™ (Life Techynolooles, Eugene Oreg.,USA). The aminoallyl-DNA (aa-DNA) is purified using QIAquick™ (Qiagen,Duesseldorf, Germany). A volume of 7 μL of sodium acetate 3 M and 400 μLbinding buffer supplied by the Kit are added. The mixture is allowed tostand 5 min according to the manufacturer's instructions forpurification. The eluted aa-DNA is concentrated in a Vacufuge™ until dryand is resuspended in 4.5 μL of sodium bicarbonate 100 mM (pH 9.0). Thereaction is conducted in darkness at room temperature overnight. Theaa-DNA labeled is purified using QIAquick™ (Qiagen, Duesseldorf,Germany) and eluted with 50 uL water. The aa-DNA labeled is quantifiedin a Nanodrop™ 1000 spectrophotometer (Thermo Fisher Scientific,Waltham, Mass., USA).

After completely drying in a Vacufuge concentrator, the aa-DNA labeledis resuspended in hybridation mixture containing: ˜0.1 optical densityunits of aa-DNA labeled from each sample; SSC (5×) buffer; 0.1% SDS; 1×buffer TE. The hybridation mixture is desaturated at 94° C. for 5 minand finally at 65° C. for 30 s. The mixture is placed in the microarrayslide previously placed in a chamber for microarray hybridization andcovered using a RNase-free plastic coverslip (Hybrislip, Schleicher &Schuell, HS40—40×22 mm; HS22—22×22 mm via Thomas Scientific). Thehybridation chamber is incubated at 42° C. overnight.

The slide is placed in a 50 mL conical tube for the first wash and avolume of 50 mL of SSC (1×) buffer and of SDS 0.05% are added for 1 min.The washing is repeated twice using a volume of 50 mL of SSC (0.06×) for2 min. The slide is centrifuged at 1500 rpm during 5 min.

The microarray slide is scanned in GenePix® 4100A Microarray Scanner(Molecular Devices, Silicon Valley, Calif., USA) at 5 μm resolution onthe channels PMT 647 and PMT 555. The data of the scanning is analyzedin GenArise v2.7 software to obtain the data of select genes that aresignificantly differentially expressed onto control and treated samples.The genes are classified into categories and assigned a numerical value«zScore» base in the data normalization. The EST, Nucleotide andUnigenes sequences from the microarray are downloaded using«Batch-Entrez» at http://www.ncbi.nlm.nih.gov/sites/batchentrez andloaded in Blast2GO as FASTA files (Conesa, A., Götz, S., Garcia-Gómez,J. M., Terol, J., Talón, M., & Robles, M. 2005. Bioinformatics, 21,3674-3676), performing a BlastX using an e-Value of 1×10³ againstNon-Redundant Database and functional annotation from gene ontologyDatabase.

7-4—Analysis of Genes by RT-qPCR

RT q-PCR is performed on the 8 samples of shrimp treated withcholine/pentaborate complex and a similar number of samples of shrimpnon-treated for the microarray analysis (n=16) in order to validate 7genes of interest (GOIs) and 3 reference genes. Primer of manganesesuperoxide dismutase (MnSOD) [Lv MnSOD q-PCR 149F (forward primer, SEQID NO: 5: GGG CTT CAT TAA CAA CCT AAT TGC), and Lv MnSOD q-PCR 149R(reverse primer, SEQ ID NO: 6: GGG CTT CAT TAA CAA CCT AAT TGC)]; andreference gene ribosomal protein L8 [Lv L8pro q-PCR 166F (forwardprimer, SEQ ID NO: 7: TAG GCA ATG TCA TCC CCA TT) and Lv L8pro q-PCR166R (reverse primer, SEQ ID NO: 8: TCC TGA AGG AAG CTT TAC ACG)] aretaken from Gomez-Anduro et al. (2006). Primer design is performed in theonline application «primer3plus» (Untergasser, A., Nijveen, H., Rao, W.,Bisseling, T., Geurts, R., Leunissen, J. A. M. 2007, Nucleic AcidsResearch, 35: 71-74) based in the following features: 18-24 nt length;GC content 35-65% product size 80-250 bp; and ≤2 GC clamp (Wang, X. andSeed, B., 2007). The primers are evaluated in the online application«Oligo Evaluator» Sigma Aldrich™ (St' Louis, Mo.) to check thermodynamiccharacteristics based in self-aligned primer and primer-dimerstructures. The primers selected are ≤−2 Kcal/mol and evaluated using anin silico PCR software «Primer Digital» (Kalender et al., 2011). RNAextracts used for the microarray hybridizations are converted to cDNAusing promega GoScript™ Reverse Transcription System (PromegaCorporation, Madison, Wis., USA) and SsoFast™ EvaGreen® Supermix(Bio-Rad, Hercules, Calif., USA) for RT-qPCR. Fluorescence is detectedby using the Rotor gene 6000 Real-Time PCR detection system (Corbette).A three-step cycling protocol is used with primer specific annealingtemperatures. The RT q-PCR cycle is 95° C. for 10 min; followed by 40cycles of 95° C. for 15 s and 20 s at the primer specific annealingtemperature (see Table 1); 72° C. for 25 s after which the plate isread. The annealing temperature range for the primers is 56-62° C. Meltcurve analysis is performed to determine product specificity over thetemperature range of 65-95° C. in 1° C. increments, and read every 5 s.The same six samples treated and non-treated shrimps (n=12) used formicroarray are assayed in triplicate for RT q-PCR, in addition totriplicates of negative RT controls and negative template controls(NTC). The geNorm software (Vandensompele et al., 2002) identified 3genes: β-actin, elongation factor 1-α and ribosomal protein L8 to be themost stable and meeting the selection criteria of M value of <1.25,representing the average expression stability and V value≤1.5,indicating pair wise variation.

The other primers are:

Ferritin: Ferritin Forward, SEQ ID NO: 9: CAAGCGAACCTCTGGAAATC, andFerritin Reverse, SEQ ID NO: 10: TGGCAAATCCAGGTAGAGC.Toll-like receptor: LvToll Forward, SEQ ID NO: 11: GCCCTAAATGATGGATGAC,and LvToll Reverse, SEQ ID NO: 12: GCCAAGGGAAAAAGAAAT.Elongation Factor 1-α: LvEF1A Forward, SEQ ID NO: 13:CCACACTGCTCACATTGC, and LvEF1A Reverse, SEQ ID NO: 14:GAAGGTCTCCACGCACAT. B-Actin: LvACTB Forward, SEQ ID NO: 15:TGGGACGACATGGAGAAG, and LvACTB Reverse, SEQ ID NO: 16:GGGGGTGTTGAAGGTCTC. Pre-amylase 1: LvPAMY Forward, SEQ ID NO: 17:CCGTCTCCTATAAACTCGTCACTC, and LvPAMY Reverse, SEQ ID NO: 18:TCGCCGTAGTTTTCAATGTTC. Trypsinogen 1: LvTry Forward, SEQ ID NO: 19:TCGTCGGAGGAACTGACG, and LvTry Reverse, SEQ ID NO: 20:TGCCCTCATCCACATCCT. Lipase Digestive 1: LvLIP Forward, SEQ ID NO: 21:TCCTGGCTCACACACCTG, and LvLIP Reverse, SEQ ID NO: 22:GTCCTTCAGCGAGCCTTG. Cathepsin L: LvCPL Forward, SEQ ID NO: 23:CGTCCTTCCAGTTCTACCAT, and LvCPL Reverse, SEQ ID NO: 24:ATCTGGATGTAGCCCTTGTT.7-5—Statistical Analysis

At termination of the exposure experiment, survival and mortality dataare analyzed using TOXSTAT probit (adapted from Stephan, 1977) analysissoftware to calculate a LC₅₀ with a 95% confidence interval. Microarraygene expression values are analyzed using a one-way ANOVA and 100permutations to detect significantly at p-value of 0.05. A Tukeypost-hoc test is also used to identify significantly differentiatedgenes affected by the choline/pentaborate complex treatment. A userdefined k-means cluster analysis (n=4) is run with a Pearson centereddistance matrix and 100 iterations using GeneSpring (Agilent,Mississauga, ON, Canada). The mean CNRQ values from the RT q-PCRanalysis of GOI for treated and non-treated shrimps are compared toidentify significant differences in relative abundance using a one-wayANOVA with multiple test corrections and significant p-value<0.05. TheCNRQ values are log2 transformed and compared to microarray logetransformed expression ratios.

7-6—Results for Analysis of Genes by RT q-PCR

7-6-1—Treatment of L. vannamei Shrimp with Choline/Pentaborate Complex

The two shrimp groups fed with a commercial feed (control group), andthe other group, fed with the same commercial feed, but supplementedwith choline/pentaborate complex at dose 5 mg/g of feed are maintainedin the bioassay laboratory for up to 28 days.

It is of interest to mention that the shrimp fed with the commercialdiet supplemented 5 mg of choline/pentaborate complex had a 20%increment in mean weight, when compared with the shrimps control group(FIG. 6). After 14 days of treatment, shrimps are sampled for the DNAmicroarray analysis, but also, to confirm that these shrimps are moreresistant against WSSV and V. parahaemolyticus infections as previouslyreported.

7-6-2—DNA Microarray Analysis of Shrimp Treated with Choline/PentaborateComplex

The shrimp DNA microarray contains 60000 spots. Table 2 summarizes theclassification of differential gene expression based in the patterns ofabsorbance source in two channels: Alexa-555 and Alexa-647 dye, and thenumber of Up- and Down-regulated genes in the microarray analysis.

TABLE 1 Primer sequences used in the RT-qPCR Align Melt NCBI Temp PeakSize Accession Primer ID Gene Name (° C.) Sequence (5′-3′) (° C.) (bp)AY955373.1 LvFerritinF Ferritin 58 CAAGCGAACCTCTGGAAATC 83.5 230LvFerritinR TGGCAAATCCAGGTAGAGC FE147224.1 LvTollF Toll-like 62GCCCTAAATGATGGATGAC 88.2 151 Receptor GCCAAGGGAAAAAGAAAT GU136229.1LvEF1AF Elongation Factor 56 CCA CACTGCTCACATTGC 85.5 151 1-α LvEF1ARGAAGGTCTCCACGCACAT JF288784.1 LvACTBF β-actin 60 TGGGACGACATGGAGAAG 86.7150 LvACTBR GGGGGTGTTGAAGGTCTC X77318.1 LvPAMYF Pre-amylase 1 58CCGTCTCCTATAAACTCGTCACTC 88.5 259 LvPAMYR TCGCCGTAGTTTTCAATGTTCJQ277721.1 LvTryF Trypsinogen 1 58 TCGTCGGAGGAACTGACG 89.2 210 LvTryRTGCCCTCATCCACATCCT FJ619564.1 LvLIPF Lipase Digestive 1 PENDINGTCCTGGCTCACACACCTG PENDING 231 LvLIPF GTCCTTCAGCGAGCCTTG X99730.1 LvCPLFCathepsin L PENDING CGTCCTTCCAGTTCTACCAT PENDING 166 LvCPLRATCTGGATGTAGCCCTTGTT

TABLE 2 Statistical analysis of the number of Up- and Down-regulatedgenes in the DNA shrimp microarray analysis Expression Levels (Score)Number of spots All Spots 60,000 Spots available to analysis 57,193Without signal 37 >2 1,650 1.5 to 2 1,984 1 to 1.5 3,662 No regulatedgenes (−1 to 1) 43,950 −1.5 to −1 3,221 −1.5 to −2 1,259 <−2 1,4347-6-3—Gene Ontology Analysis

Gene ontology (GO) is commonly used to categorize gene products andstandardize their representation across species. In order to eliminateredundancy only the 2-fold Up- and Down-regulated differentiallyexpressed genes are submitted to Blast2GO suite for the assignment ofseveral functional groups based on GO terminology. There were 583Up-regulated genes in samples from shrimp fed for 14 days with the feedsupplemented with choline/pentaborate complex at dose 5 mg/g, expressedgenes fall into the followed biological processes:

-   -   1. Cellular Processes (20%);    -   2. Metabolic Process (18%);    -   3. Single-organism Process (17%);    -   4. Biological Regulation (9%);    -   5. Cellular Component Organization or Biogenesis (7%);    -   6. Localization (7%);    -   7. Response to Stimulus (6%);    -   8. Multicellular Organismal Process (6%);    -   9. Developmental Process (6%) and Signaling (4%) (FIG. 7).

The Molecular Functions of the 583 for 2-fold Up-regulated expressedgenes fall into four categories:

-   -   10. Binding Activity (44%);    -   11. Catalytic Activity (43%);    -   12. Transporter Activity (7%);    -   13. Structural Molecule Activity (6%) (FIG. 8).

The Fisher's exact test from GO terms of the 2-fold Up-regulated genes(shrimps treated with choline/pentaborate complex) versus all genes(shrimp control group) from DNA microarray shows thatcholine/pentaborate complex treated shrimps have gene with increasedexpression in the categories described as follows:

-   -   14. Ion Transport (8.15%);    -   15. Monovalent Inorganic Cation Transport (5.92%);    -   16. Cytoskeletal Protein Binding (5.55%);    -   17. Hydrogen Transport (4.81%);    -   18. Proton Transport (4.81%);    -   19. Actin Cytoskeleton (4.62%);    -   20. Calcium Ion Binding (4.25%);    -   21. Monosaccharide Metabolic Process (4.10%);    -   22. Positive Regulation of Biosynthetic Process (3.70%);    -   23. Positive Regulation of Cellular Biosynthetic Process (3.70%)        (FIG. 9).

Regarding the Functional Annotation of the 2-fold Down-regulatedexpressed genes from the DNA microarray of the Biological Processes, itis found that there were 262 genes with a 2-fold Down-regulatedexpression in shrimp fed for 14 days with the feed supplemented withcholine/pentaborate complex 5 mg/g, expressed genes fall into thefollowed Biological Processes:

-   -   24. Cellular Process (20%);    -   25. Metabolic Process (20%);    -   26. Response to Stimulus (5%);    -   27. Single-organism Process (17%);    -   28. Multicellular Process (6%);    -   29. Signaling (3%);    -   30. Localization (8%);    -   31. Biological Regulation (9%);    -   32. Developmental Process (5%);    -   33. Cellular Component Organization or Biogenesis (7%) (FIG.        10).

The Molecular Function of the 262 genes in the 2-fold Down-regulatedexpressed genes fall into four categories:

-   -   34. Binding (44%);    -   35. Catalytic Activity (40%);    -   36. Transporter Activity (9%);    -   37. Structural Molecule Activity (4%);    -   38. Enzyme Regulator Activity (3%) (FIG. 11).

The Fishers exact test from GO terms of the 2-fold Down-regulated genes(shrimps treated with choline/pentaborate complex) versus all genes(shrimp control group) from DNA microarray showed that shrimp treatedwith choline/pentaborate complex over-represented gene categoriesdescribed as follows:

-   -   1. Regulation of protein metabolic process (10.56%);    -   2. Transporter activity (10.56%), extracellular region (9.75%);    -   3. Extracellular region part (5.69%), extracellular space        (4.87%);    -   4. Translation factor activity, nucleic acid binding (4.47%);    -   5. Photoreceptor cell differentiation (2.43%);    -   6. Gas transport (2.03%);    -   7. Oxygen transporter activity (2.03%);    -   8. Starch metabolic process (2.03%);    -   9. Sucrose metabolic process (2.03%);    -   10. Toll-like Receptor for signaling pathway (1.62%) (FIG. 12).        7-6-4—Comparative Analysis of Metabolic Pathways

The L. vannamei transcriptomic sequences from the shrimp DNA microarrayare compared to ESTs and nucleotide sequences from Drosophila present inthe NCBI database in order to detect the presence of proteins that areover-expressed in different Metabolic Pathways. The results of the2-fold Up-regulated genes, related to genes and enzyme expressed intoeach group are summarized in Table 3. Similarly, the results of the2-fold Down-regulated genes, related to genes and enzyme expressed intoeach group are summarized in Table 4.

TABLE 3 Metabolic pathways of DNA shrimp microarray analysis of the 2-Upregulated genes in L. vannamei treated with the choline/pentaboratecomplex supplemented (5 mg/g) commercial feed. Metabolic Pathway # Genes# Enzymes Oxidative phosphorylation 18 6 Purine metabolism 14 7Glycolysis 8 8 Amino sugar and nucleotide Sugar 8 7 metabolismGlutathione metabolism 7 5 Pyruvate metabolism 6 6 Valine, leucine andisoleucine 6 6 degradation Cytochrome P450 6 3 Pentose phosphate pathway5 4

TABLE 4 Metabolic pathways of DNA shrimp microarray analysis of the2-Down regulated genes in L. vannamei treated with thecholine/pentaborate supplemented (5 mg/g) commercial feed MetabolicPathway # Genes # Enzymes Oxidative phosphorylation 6 4 Pentosephosphate pathway 5 5 Starch and sucrose metabolism 5 2 Purinemetabolism 4 3 Phenylpropanoid biosynthesis 3 1 Aminoacyl-tRNAbiosynthesis 3 3 Glycolysis/Gluconeogenesis 3 3 Pentose and glucuronate3 2 interconversions Phenylalanine metabolism 3 1 Ascorbate and aldaratemetabolism 3 2

7-6-5—Data Mining of the Immune Related Genes

A search of the DNA microarray analysis, with GO term: 0006955 (immuneresponse), revealed the presence of an elevated number of relevantmolecules for the immune response that are over-expressed after shrimpare fed for 14 days with the feed supplemented with choline/pentaboratecomplex 5 mg/g. The main components related to immune response in L.vannamei that are 2-fold Up-regulated are described in Table 5.

7-6-6—Validation of Specific Genes by RT-VCR

Selected genes related to immune and digestive proteins presented in theshrimp DNA microarray (Table 1) are further validated by RT q-PCR, andthe results are illustrated in the FIG. 13.

TABLE 5 Data mining of immune related genes from the 2 Up-regulated inthe shrimp DNA Microarray # NCBIID Protein Description 1 40958211interleukin Enhancer Binding Factor 2 52863030 Probable ProteinBrick1-like 3 171595417 Actin-related Protein 3 Isoform X2 4 171649044Protein Toll 5 171484743 Srsf Protein Kinase 2 6 171604948 Profilin 7171640969 Histone Acetyltransferase P300-Partial 8 171533428 Protein Red9 171638050 Serine Threonine-protein Phosphatase 2a 65 Kda RegulatorySubunit A Alpha Isoform-like 10 171601498 Ubiquitin-40s RibosomalProtein S27a 11 171578425 Ubiquitin Family Protein 12 171527123 ProteinLsm14 Homolog A isoform X2 13 171576197 Beta--Glucan-binding Protein 14171497789 Sam Domain And Hd Domain-containing Protein 1 15 171616000Cathepsin C 16 171660130 Gluaosidase 2 Subunit Beta 17 171644991Calmodulin 18 171655889 Cytoplasmic Partial 19 171508897 DipeptidylPeptidase 1 20 171534712 Profilin 21 171606038 Dna-directed RnaPolymerases And III Subunit Rpabc6 22 171616286 Dna-directed RnaPolymerase III Subunit Rpc6 23 171649684 ExocystComplex Component2-like24 171524287 Ubiquitin-40s Ribosomal Protein S27a 25 171488738Ubiquitin-conjugating Enzyme E2 26 171580467 Ubiquitin C 27 171533219Lrr And Pyd Domains-containing Protein 10 28 171626089 A Chain OrallyActive 2-amino Thionopyrimidine Inhibitors Of Tho Hsp90 Chaperone 29156637483 Lipopolysaccharide And Beta-glucan Binding Protein 30 89258160Ecdysteroid-regulated Protein 31 68271149 Dead Box Helicase Partial 321907112 Bone Morphogenetic Protein 6 33 29838465 Beta-glucan-bindingProtein

Globally, commercial shrimp diets supplemented with 5 mgcholine/pentaborate complex by gram of feed are effective to stimulatethe non-specific immune system of shrimp and to improve naturalresistance of L. vannamei to WSSV and Vibrio parahaemolyticusinfections.

Choline/pentaborate complex supplementation can increase the immunologicreactivity of shrimps as revealed by the number of immune related genesexpressed in the DNA shrimp microarray analysis. There are at least 33immune related genes that are over-expressed (Table 5). Some of thesegenes are further validated by RT q-PCR, and our results revealed thatthe expression of some of these genes, such as the Toll-Like Receptorand SOD increased more than 200 fold (FIG. 13), clearly indicating theimmunostimulatory activity of choline/pentaborate complexsupplementation of a commercial shrimp diet.

As arthropod species, shrimp mainly rely on the innate immune system,which consists of humoral and cellular responses against viralinfections. The direct or indirect recognition of pathogens orpathogen-associated molecular patterns by germ line-encoded proteinscalled pattern recognition receptors (PRRs) that tightly related toToll-like Receptors leads to rapid humoral and cellular immune responses(Li, F., Xiang, J. 2013, Dev. Comp. Immunol. 39, 11-26).

It is of interest to mention that invertebrates do not possess anadaptive immune system based on highly specific antibodies and antigenreceptors. They must rely on efficient immune defense to protect themagainst invaders. It has been proven that hemocytes are key cells forinnate invertebrate defense reactions. One important immune defensereaction of crustacean hemocytes is phagocytosis when the organism isattacked by microorganisms or viruses. During the course ofphagocytosis, the host oxidases (e.g. NADPH oxidase, and particularlyprophenol oxidase that is involved in the shrimp clotting system and inthe innate immunity) get activated which in turn enhances the glycolyticreactions (explaining amylase gene over-expression) that will increasethe consumption of oxygen, and induce the production of a mass ofreactive oxygen species (ROS) such as superoxide anion (O₂ ^(⋅)  ),hydrogen peroxide (H₂O₂) and hydroxyl radical (OH^(⋅)). Though ROS cankill foreign invaders, the mass accumulation of these reactive moleculesin animals can cause serious cell damage. WSSV infection can cause therelease of ROS and increase the oxidative stress in shrimp and leads toa high level of lipid peroxidation. Consequently, the rapid eliminationof these excessive ROS is essential for the proper functioning of cellsand the survival of organisms. This is performed by antioxidant enzymesincluding superoxide dismutases (SOD) that scavenges the superoxideanions. SOD detoxifies superoxide radicals by converting them tohydrogen peroxide and oxygen. Hydrogen peroxide is then transformed towater and oxygen by catalase or other antioxidant compounds, providinginnocuous compounds to the cell.

Besides over-expression of immune related genes in shrimp treated withdiets supplemented with choline/pentaborate complex, other biologicalprocesses are modulated too in L. vannamei, including the expression ofgenes associated to: Response to stimuli (43), Metabolic process (166),Cellular process (159), Biogenesis (59), Developmental process (40),Biological regulation (73) Localization (66), and Signaling (26) (FIG.10, Table 3).

Shrimp diets supplemented with choline/pentaborate complex atconcentration of 5 mg/g of feed are effective in reducing the impact ofWSSV infection when following standardized administration protocols.This represents a major breakthrough for the control of a disease thataffects a significant number of commercial shrimp production operations.The inclusion of choline/pentaborate complex used as additive incommercial diets provides an efficient mechanism for stimulation of theshrimp immune system that contribute to improve natural resistance of L.vannamei to WSSV and Vibrio parahaemolyticus infections.

EXAMPLE-8 Other Quaternary Ammonium Compounds Used to Complex with BoricAcid or its Derivatives

8-1—Complexation of (2-Hydroxyethyl)Triethylammonium with Boric Acid (orits Derivatives)

The complexation of (2-hydroxyethyl)triethylammonium and boric acid (orits derivatives) is prepared under identical conditions as describedpreviously in EXAMPLE 1, except that (2-hydroxyethyl)triethylammonium isused, instead of (2-hydroxyethyl)trimethylammonium (FIG. 14-A)

8-2—Complexation of (2,3-Dihydroxyethyl)Triethylammonium with Boric Acid(or its Derivatives)

Since choline ([2-hydroxyethyl]trimethyl ammonium) possesses a hydroxylgroup, the complexation could be less stable, because there is ahydroxyl groups involved in formation of the complex with boric acid (orits derivatives). Consequently, the use of choline having two hydroxylgroups such as (2,3-dihydroxypropyl)trimethylammonium chloride seemsmore stable probably due to its contribution of two hydroxyl groups incomplexing with boric acid (or its derivatives).

The complexation of choline and boric acid is prepared under identicalconditions as described previously in EXAMPLE 1, except that(2,3-dihydroxypropyl) trimethylammonium is used, instead of(2-hydroxyethyl)trimethylammonium (FIG. 14-B).

8-3—Complexation of Choline with Phenylboronic Acid

The complexation of choline and phenylboronic acid is prepared underidentical conditions as described previously in EXAMPLE 1, except thatphenylboronic acid is used, instead of boric acid (FIG. 15) and thevolume of distilled water is 800 mL, instead of 400 mL.

8-4—Complexation of Choline with Myristylboronic Acid

The complexation of choline and phenylboronic acid is prepared underidentical conditions as described previously for «Complexation ofcholine with phenylboronic acid», except that Myristylboronic acid isused, instead of phenylboronic acid (FIG. 16).

While preferred embodiments have been described above and illustrated inthe accompanying drawings, it will be evident to those skilled in theart that modifications may be made without departing from thisdisclosure. Such modifications are considered as possible variantscomprised in the scope of the disclosure.

The invention claimed is:
 1. An ion of formula I, or a pharmaceuticallyacceptable salts thereof, and stereoisomers thereof:

wherein R¹ is C₁₋₆ alkylene, R², R³, and R⁴ are independently C₁₋₆ alkyloptionally substituted with one or more —OH, and R⁵ is selected from—O—B(OH)₂,

with the proviso that when R¹ is —CH₂—CH₂— and R², R³, and R⁴ are —CH₃,R⁵ is different than —O—B(OH)₂.
 2. The ion of claim 1, or apharmaceutically acceptable salts thereof, and stereoisomers thereof,wherein R¹ is —CH₂—CH₂—.
 3. The ion of claim 1, or a pharmaceuticallyacceptable salts thereof, and stereoisomers thereof, wherein any one ofR², R³, and R⁴ is independently —CH₃, —CH₂—CH₃—, or —CH₂—CH₂—CH₃.
 4. Theion of claim 1, or a pharmaceutically acceptable salt thereof, whereinR¹ is —CH₂—CH₂—, and R², R³, and R⁴ is independently —CH₃.
 5. The ion ofclaim 1, or a pharmaceutically acceptable salt thereof, wherein said ionis selected from the following ions:

or a combination thereof.
 6. A mixture of the ion of claim 1, or apharmaceutically acceptable salt thereof, comprising the following-ionsof formula I,:

in combination with the following ion


7. A pharmaceutical composition comprising an ion of any one of claims 1to 6, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 8. The pharmaceutical compositionof claim 7, for use in the treatment of a pathogenic infection in asubject.
 9. The pharmaceutical composition of claim 7, for use in thetreatment of a pathogenic infection in a crustacean in need thereof. 10.The pharmaceutical composition of claim 7, wherein said subject isselected from the group consisting of a mammal, a fish, a bird, and acrustacean.
 11. The pharmaceutical composition of claim 10, wherein:said mammal is selected from the group consisting of a human, a bovine,an equine, and an ungulate; said fish is selected from the groupconsisting of a hagfish, a lamprey, a cartilaginous fish and a bonyfish; said bird is selected from the group consisting of chicken, aturkey, and a fowl; and said crustacean is selected from the groupconsisting of a shrimp, a crab, a lobster, a langouste.
 12. Thepharmaceutical composition of claim 8, wherein said pathogenic infectionis caused by a virus, a microorganism, or combinations thereof.
 13. Thepharmaceutical composition of claim 12, wherein: said virus is one ofmore of White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV),Yellow Head Virus (YHV), Infectious Hypodermal and HaematopoieticNecrosis (IHHNV), Spherical Baculovirus, Spawner-isolated MortalityVirus Disease, Spring Viremia of Carp (SVC caused by Rhabdoviruses), KoiHerpes Virus (KHV), Large Mouth Bass Virus (LMBV), and Baculoviruspenaei (BP); and said microorganism is one or more of Vibrioparahaemolyticus, Vibrio harveyi, V. splendidus, V. parahaemolyticus, V.alginolyticus, V. anguillarum, V. vulnificus, V. campbelli, V. fischeri,V. damsella, V. pelagicus, V. orientalis, V. ordalii, V. mediterrani, V.logei, an Enterobacteriacae, such as an Escheria coli, a Salmonella, aShigella; Clostridium botulinum, Listeria monocytogenes.
 14. Thepharmaceutical composition of claim 7, wherein said pharmaceuticalcomposition is a dietary composition.
 15. A method of treating orpreventing a pathogenic infection in a fish or a crustacean in needthereof comprising: administering a therapeutically effective amount anion of claim 1, or a pharmaceutically acceptable salt thereof.
 16. Themethod of claim 15, wherein said crustacean is selected from the groupconsisting of a shrimp, a crab, a lobster, and a langouste, and whereinsaid fish is selected from the group consisting of fish a hagfish, alamprey, a cartilaginous fish and a bony fish.
 17. The method of claim15, wherein said pathogenic infection is caused by a virus, amicroorganism, or combinations thereof.
 18. The method of 17, whereinsaid virus is one of more of White Spot Syndrome Virus (WSSV), TauraSyndrome Virus (TSV), Yellow Head Virus (YHV), Infectious Hypodermal andHaematopoietic Necrosis (IHHNV), Spherical Baculovirus, Spawner-isolatedMortality Virus Disease, Spring Viremia of Carp (SVC caused byRhabdoviruses), Koi Herpes Virus (KHV), Large Mouth Bass Virus (LMBV),and Baculovirus penaei (BP) and said microorganism is one or more ofVibrio parahaemolyticus, Vibrio harveyi, V. splendidus, V.parahaemolyticus, V. alginolyticus, V. anguillarum, V. vulnificus, V.campbelli, V. fischeri, V. damsella, V. pelagicus, V. orientalis, V.ordalii, V. mediterrani, V. logei, an Enterobacteriacae.
 19. The methodof claim 18, wherein said Enterobacteriacae is one or more of anEscheria coli, a Salmonella, a Shigella.
 20. The method of claim 15,wherein administering is by feeding said ion, or said pharmaceuticallyacceptable salt thereof, to said crustacean.